title: Cloning and expression of the 75 kDa DnaK-like protein of Chlamydia psittaci and the evaluation of the recombinant protein by immunoblotting and indirect ELISA.
abstract: The 75 kDa dnaK-like gene of Chlamydia psittaci ovine abortion strain S26/3 was isolated from an EMBL 3 chlamydial DNA library. A 7 kb DNA fragment containing the gene was subcloned into Bluescribe (M13+) plasmid and used to transform competent E. coli. These cells were found to express a cytoplasmic protein of 75 kDa. Monospecific antibodies against the protein prepared by antibody elution reacted with the native 75 kDa protein. Recombinant clones did not adhere to McCoy cell monolayers in cell adhesion studies. The 75 kDa protein purified by ion-exchange chromatography was used in immunoblotting and indirect enzyme-linked immunosorbant assay (ELISA) studies using sera previously screened for chlamydial antibodies by an indirect ELISA incorporating solubilised chlamydial elementary bodies and by microimmunofluorescence. Immunoblotting identified 6/11 sera from infected ewes that had either typical placental lesions or had been found positive on examination of stained placental smears and 1/11 sera from ewes that had no typical placental lesions. The ELISA gave positive reactions with 29 of 65 known positive sera and 15 of the 76 negative sera. It is concluded that the 75 kDa DnaK-like protein is unsuitable as an antigen for antibody detection but its potential as a component for a sub-unit vaccine against ovine enzootic abortion warrants further study.
authors.affiliation: Moredun Research Institute, Edinburgh, Scotland, UK. andei@mri.sari.ac.uk
authors.forename: I E
authors.initials: IE
authors.lastname: Anderson
authors.forename: J K
authors.initials: JK
authors.lastname: Craggs
authors.forename: S
authors.initials: S
authors.lastname: Dunbar
authors.forename: A J
authors.initials: AJ
authors.lastname: Herring
mesh_terms: D000034:Abortion, Veterinary / Q000382:microbiology; D000818:Animals; D000907:Antibodies, Bacterial / Q000032:analysis; D002690:Chlamydia Infections / Q000175:diagnosis / Q000662:veterinary; D002691:Chlamydophila psittaci; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005260:Female; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000276:immunology; D015151:Immunoblotting / Q000379:methods; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology; D012044:Regression Analysis; D012756:Sheep; D012757:Sheep Diseases
chemical_list: D000907:Antibodies, Bacterial; D017931:DNA Primers; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D011994:Recombinant Proteins; C063842:dnaK protein, E coli